Evolved orthogonal ribosomes

ABSTRACT

There is provided a method for evolving an orthogonal rRNA molecule, comprising the steps of: providing one or more libraries of mutant orthogonal rRNA molecules and introducing the libraries into cells such that the orthogonal rRNA is incorporated into ribosomes to provide orthogonal ribosomes; providing one or more orthogonal mRNA molecules which (i) are not translated by natural ribosomes, and (ii) comprise one or more orthogonal mRNA codons; assaying the translation of the orthogonal mRNA and selecting the orthogonal rRNA molecules which translate the orthogonal mRNA, wherein the assay in step (c) requires translation of one or more orthogonal mRNA codons in the orthogonal mRNA; and orthogonal ribosomes incorporating such rRNA molecules.

The present application is filed pursuant to 35 U.S.C. 371 as a U.S.National Phase application of International Patent Application No.PCT/GB07/04562, which was filed Nov. 28, 2007, claiming the benefit ofpriority to British Patent Application No. 0623974.3, which was filed onNov. 30, 2006, U.S. Patent Application No. 60/939,906, which was filedon May 24, 2007, and British Patent Application No. 0710094.4, which wasfiled on May 25, 2007. The entire text of the aforementioned.PCT/GB07/04562 is incorporated herein by reference in its entirety.

The present invention relates to orthogonal ribosomes with enhancedtranslation efficiency for orthogonal RNA. In particular, the inventionprovides orthogonal ribosomes with enhanced translation efficiency forquadruplet and amber codons.

INTRODUCTION

We recently created orthogonal ribosome-mRNA pairs that operate inparallel with, but independent of, the cellular ribosome²². Theorthogonal mRNA contains a ribosome-binding site that does not directtranslation by endogenous ribosomes but is efficiently translated by anorthogonal ribosome, which does not appreciably translate cellularmRNAs. In previous work we have explored the use of orthogonal ribosomesto create new modes of system-level translational regulation^(22, 23),and to understand the relationship between ribosome structure andfunction²⁴.

The triplet nature of the genetic code¹ is conserved across all knownorganisms. Rare exceptions to the correspondence between an mRNA tripletsequence and the amino acid encoded include frame shifts (+1, +2, −1,−2), hops, and read-through of stop signals^(2, 3). While manyprogrammed changes of reading frame require upstream sequences in themRNA that recruit additional translation factors or that interact withand prepare the translational machinery for a change of reading frame,+1 frame shift mutations that create quadruplet codons can be read,independent of upstream signals, by tRNAs with extended anticodonloops⁴⁻⁹. The apparent simplicity and uniqueness of quadruplet codons asamino acid insertion signals has led to the use of extended anticodontRNAs to encode the incorporation of unnatural amino acids, albeit withlow efficiency, both in vitro¹⁰⁻¹³ and in vivo^(12, 14, 15).

The mechanism of quadruplet decoding, the efficiency of quadrupletdecoding, and the range of quadruplet codon/anticodon pairs accessibleto the natural translational machinery have been investigated for over35 years^(4-6, 16-19). Recently, Schultz and coworkers explored thescope of quadruplet codon-anticodon pairs that operate with the naturaltranslational machinery by crossing a library of extended anticodon tRNAmutants, derived from tRNAser2, with a library of quadruplet codons⁸.They discovered a group of extended anticodon tRNAs able to read theircognate quadruplet codons, including UAGA, AGGA and CCCU, in vivo. Thein vivo efficiency of quadruplet decoding by extended anticodon tRNAs ispoor (from less than 1% to about 20%)⁶. It is clear that the efficiencyof decoding quadruplet codons with extended anticodon tRNAs is limitedby the natural ribosome, that has evolved to read a triplet code.

Evolving and engineering the natural ribosome is a challenge on severallevels. First, the ribosome is massive: at 2.5 MDa and containing threelarge RNAs and 52 proteins, it is an order of magnitude larger than mostmacromolecules that have been engineered or evolved. Fortunately,structural biology, biochemistry and mutagenesis have begun to provideinsights into the molecular basis of ribosome function²⁰, and provide amolecular basis for targeted efforts to expand ribosome function.Second, the ribosome is essential, and highly conserved. Many mutationsin ribosomal components are dominant negative, deleterious or lethalsince they compromise the efficient and accurate synthesis of theproteome²¹.

The genetic code of prokaryotic and eukaryotic organisms has beenexpanded to allow the in vivo, site-specific incorporation of over 20designer unnatural amino acids in response to the amber stop codon. Thissynthetic genetic code expansion is accomplished by endowing organismswith evolved orthogonal aminoacyl-tRNA synthetase/tRNA_(CUA) pairs thatdirect the site-specific incorporation of an unnatural amino acid inresponse to an amber codon. The orthogonal aminoacyl-tRNA synthetaseaminoacylates a cognate orthogonal tRNA, but no other cellular tRNAs,with an unnatural amino acid, and the orthogonal tRNA is a substrate forthe orthogonal synthetase but is not substantially aminoacylated by anyendogenous aminoacyl-tRNA synthetase. Genetic code expansion in E. coliusing evolved variants of the orthogonal Methanococcus jannaschiityrosyl-tRNA synthetase/tRNA_(CUA) pair greatly increases unnaturalamino acid-containing protein yield since, in contrast to methods thatrely on the addition of stoichiometrically pre-aminoacylated suppressortRNAs to cells or to in vitro translation reactions, the orthogonaltRNA_(CUA) is catalytically re-acylated by its cognate aminoacyl-tRNAsynthetase enzyme, thus aminoacylation need not limit translationalefficiency.

While in vivo genetic code expansion is clearly a major advance, theefficiency of site-specific unnatural amino acid incorporation in E.coli via amber suppression is severely limited; Release factor-1 (RF-1)mediated peptide chain termination competes with tRNACUA mediated chainelongation, and therefore 70-80% of polypeptide synthesis initiated ongenes containing a single amber stop codon is terminated at that codon.This clearly limits (to ˜20-30%) the efficiency with which proteinscontaining unnatural amino acids are synthesized from genes containing asingle internal amber stop codon. Moreover, the efficiency of unnaturalamino acid incorporation decreases drastically with increasing amberstop codons in a gene, such that less than one-tenth of proteinsynthesis initiated on a gene containing two amber codons typicallyreaches completion.

Many potential applications of unnatural amino acid mutagenesis,including the translational incorporation of amino acids correspondingto post-translational modifications present at multiple sites inproteins (eg: methylation, acetylation, phosphorylation), require moreefficient methods of incorporation to make useful amounts of protein.Moreover the introduction of biophysical probes and chemically preciseperturbations into proteins in their native cellular context offers theexciting possibility of understanding and controlling cellular functionsin ways not previously possible. However, the large amount of truncatedprotein produced in these experiments may provide a substantialperturbation to precisely the system under study, confounding meaningfulconclusions about the function of full-length unnatural aminoacid-containing protein in the cell.

Unnatural amino acid incorporation in in vitro translation reactions canbe increased by using S30 extracts containing a thermally inactivatedmutant of RF-1. Unfortunately, though temperature sensitive mutants ofRF-1 allow transient increases in global amber suppression in vivo, RF-1knockouts are lethal, and are therefore not a viable option for stablyand specifically increasing the efficiency of unnatural amino acidincorporation in E. coli. Increases in tRNACUA gene copy number and atransition from minimal to rich media have provided some improvement inthe yield of proteins incorporating an unnatural amino acid in E. coli,but the efficiency of unnatural amino acid incorporation (defined as theratio of full length protein to truncated protein) is still only 20-30%.Additional improvements possible through a further increase in tRNA copynumber are problematic for several reasons. First, this strategyincreases the extent to which natural aminoacyl-tRNA synthetasesaminoacylate the orthogonal tRNA, potentially leading to natural aminoacid incorporation in response to the amber, codon. Second, the plasmidencoded tRNACUA gene repeats typically used to achieve increases in tRNAcopy number are prone to recombination-mediated inactivation. Third, thestrategy indiscriminately increases suppression of all amber codons inthe cell and therefore enhances the read-through of stop codons onchromosomal genes (320 genes in E. coli terminate in UAG, including 44essential genes); this strategy will therefore interfere with cellularprotein synthesis and potentially disturb cellular physiology.

Despite these disadvantages, we have successfully developed anorthogonal ribosome which reads orthogonal mRNA codons with enhancedefficiency compared to natural ribosomes.

SUMMARY OF THE INVENTION

Unlike the progenitor ribosome in natural cells, orthogonal ribosomesare not responsible for synthesizing the proteome, and it is thereforebe possible to further diverge their function. However, this possibilityhad not been realised in the prior art. We have now demonstrated thefirst example of synthetic evolution of ribosome function in livingcells. We have shown that orthogonal ribosomes can be evolved to decodemore efficiently a range of extended codons using tRNAs with extendedanticodon loops. The evolved orthogonal ribosome, ribo-X, preferentiallyreads quadruplet codons with extended anticodon tRNAs and can showspecificity for Watson-Crick base pairs at the fourth position of thecodon-anticodon interaction. Ribo-X also improves amber suppression byamber suppressor tRNAs. Finally we have provided experimental supportfor a model which explains the mode of action of ribo-X, and implicatesthe 530 loop, in the ribosome decoding centre, in functionalinteractions with RF1.

In a first aspect of the present invention, therefore, there is providedan evolved orthogonal ribosomal RNA which possesses an enhancedefficiency of tRNA-dependent reading of orthogonal mRNA codons.

The rRNA of the invention differs from orthogonal rRNA (O-rRNA)molecules of the prior art, in that it not only shows specificity fororthogonal mRNA (O-mRNA), it shows an improved efficiency of translationof orthogonal mRNA codons compared to known orthogonal or naturalribosomes.

As noted above, although the prior art does document the reading ofquadruplet codons by natural ribosomes, the efficiency of such readingis at most 20% of the efficiency with which triplet codons are read. Incontrast, evolved O-ribosomes according to the invention are able todecode O-codons, such as quadruplet codons, approximately 10 times moreefficiently than the same O-ribosomes can decode natural triplet codons.Moreover, they are more efficient at decoding quadruplet codons thannon-evolved natural or O-ribosomes.

An orthogonal RNA codon, as used herein, is a codon which does notencode one of the 20 natural amino acids in the natural genetic code.Unnatural amino acids have been incorporated into proteins usingmodified tRNA which is charged with a unnatural amino acid, in place ofthe natural tRNA; however, this has previously been achieved usingnatural codons, both changing their specificity though modification oftRNA. By evolving a ribosome to decode orthogonal codons, such asextended codons, more efficiently, we have taken a different approachand developed an artificial alternative to the natural genetic code.Moreover, we have improved the efficiency of incorporation of aminoacids into polypeptides using orthogonal tRNA, improving the efficiencyof production of proteins incorporating natural and/or non-natural aminoacids using mRNA with an artificial genetic code.

Increased efficiency can be measured, for example, by comparing therelative concentrations of an antibiotic to which resistance isconferred by an O-mRNA encoding an antibiotic resistance gene. Forexample, an evolved O-ribosome according to the invention is able toconfer resistance to chloramphenicol acetyltransferase at aconcentration 10 times higher when translating a mRNA comprising anorthogonal codon than when translating a mRNA comprising only tripletcodons.

Preferably, the orthogonal mRNA codons are extended codons or stopcodons. Advantageously, the orthogonal mRNA codon is a quintuplet codon,a quadruplet codon or an amber stop codon.

Preferably the orthogonal mRNA comprises one or more amber stop codons,preferably two or more amber stop codons, preferably three or more amberstop codons, preferably four or more, amber stop codons, preferably fiveor more amber stop codons, preferably six or more amber stop codons,preferably seven or more amber stop codons, preferably ten or more amberstop codons, or even more. The advantage of these embodiment(s) is thatin the prior art, multiple amber stop codons lead to a dramaticreduction in translation efficiency, whereas according to the presentinvention these mRNAs are translated at greatly enhanced efficiency.This advantage is correspondingly greater, the greater the number ofamber stop codons present in the mRNA of interest.

The orthogonal rRNA of the present invention is preferably a 16S rRNA.16S rRNA forms the A-site in the ribosome and is responsible for bindingof the extended anticodon tRNA to the ribosome.

Preferably, the orthogonal rRNA of the invention is a mutated 16S rRNA.The 530 loop of 16S rRNA is proximal to the codon-anticodon helix;preferably, the mutated 16S rRNA is mutated in the 530 loop, betweenpositions 529 and 535.

Preferably, the orthogonal 16S rRNA comprises A531G and U534A mutations.Advantageously this orthogonal 16S rRNA is incorporated into a mutantribosome, which possesses A531G and U534A mutations, referred to hereinas ribo-X.

According to a second aspect of the invention, there is provided amethod for evolving an orthogonal rRNA molecule, comprising the stepsof:

-   -   (a) providing one or more libraries of mutant orthogonal rRNA        molecules and introducing the libraries into cells such that the        orthogonal rRNA is incorporated into ribosomes to provide        orthogonal ribosomes;    -   (b) providing one or more orthogonal mRNA molecules which (i)        are not translated by natural ribosomes, and (ii) comprise one        or more orthogonal mRNA codons;    -   (c) assaying the translation of the of the orthogonal mRNA and        selecting the orthogonal rRNA molecules which translate the        orthogonal mRNA,    -   wherein the assay in step (c) requires translation of one or        more orthogonal mRNA codons in the orthogonal mRNA.

Preferably, the libraries of orthogonal rRNA molecules are libraries of16S rRNA molecules, advantageously mutated in the 530 loop, betweenpositions 529 and 535.

Preferably, the libraries of orthogonal rRNA molecules comprise A531Gand U534A mutations.

Preferably, the orthogonal mRNA encodes a selectable marker; this markermay, for example, promote cell survival. Examples of selectable markersinclude chloramphenicol acetyltransferase, which allows the cell tosurvive exposure to chloramphenicol; cells which express CAT are thusselectable over cells that do not or do so less efficiently.

The orthogonal rRNA of the invention is useful in a variety oftranslation systems. For example, it can be used to improve systemswhich incorporate unnatural amino acids into cells. Accordingly, theinvention provides a method for incorporating a unnatural amino acidinto a polypeptide, comprising the steps of:

-   -   (a) providing a mRNA molecule comprising a stop codon in a        reading frame thereof, wherein read-though of the stop coding        can result in the incorporation of a unnatural amino acid;    -   (b) providing an orthogonal rRNA molecule according to any one        of the claims, which reads the stop codon more efficiently than        natural rRNA; and    -   (c) using a suppressor tRNA to incorporate a unnatural amino        acid into the polypeptide encoded by the mRNA molecule.

Preferably, the stop codon is a UAG amber stop codon; advantageously,the orthogonal rRNA molecule is an orthogonal rRNA molecule according tothe first aspect of the invention.

In a further aspect, there is provided a cell comprising two or moreprotein translation mechanisms, wherein:

-   -   (a) a first mechanism is the natural translation mechanism        wherein mRNA is translated by a ribosome in accordance with the        natural genetic code;    -   (b) a second mechanism is an artificial mechanism, in which        orthogonal mRNA comprising orthogonal codons is translated by an        orthogonal ribosome;    -   wherein the orthogonal codons in the orthogonal mRNA are        (i) not translated by the natural ribosome, or        (ii) translated less efficiently by the natural ribosome than by        the orthogonal ribosome, or        (iii) translated into different polypeptides by the orthogonal        ribosome and the natural ribosome.

The orthogonal codons are preferably extended codons, and advantageouslyquadruplet codons.

Alternatively, the orthogonal codons are stop codons, such as amber stopcodons.

Preferably, the orthogonal ribosome in the cell incorporates anorthogonal rRNA according to the first aspect of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Design of ribosome decoding libraries. A. Structure of a tRNAanticodon stem loop (yellow) bound to mRNA (purple) in the A site of theribosome (green). The 530 loop is shown in orange. B. Secondarystructure of the 530 loop. The boxed sequences form a pseudo knot, ψ ispseudouridine and m7G is 7-methyl guanosine. C. The sequence of ribosomedecoding libraries. 3D structure figures were created using Pymol v0.99and PDB ID 1IBM.

FIG. 2: Selection of orthogonal ribosomes for improved decoding ofquadruplet codons. A. Schematic of the selection. Quadruplet codons onan orthogonal chloramphenicol acetyl transferase mRNA (purple) aredecoded by orthogonal ribosomes (green) using extended anticodon tRNAs(yellow). The fidelity of incorporation is ensured by using serineinserting tRNAs and quadruplet codons at sites in the protein thatrequire serine. Cells containing ribosome library members with improvedquadruplet decoding are selected on chloramphenicol.

B. The anticodon stem loop sequences of the UAGA and UCCU decoding tRNAsused. C. The sequence surrounding the quadruplet codons in O-cat. Thevariable quadruplet codons “Quad” are denoted “nnnn”. The O-frame UAGAsequence shows the mutations made to test the competition betweenquadruplet and triplet decoding.

FIG. 3. Selection and characterization of ribosomes with enhanced tRNAdependent decoding of quadruplet codons A. The selection of ribosomesthat decode UAGA and AGGA codons, using cognate tRNAs derived fromtRNAser2. The top sequence trace shows the 16S rDNA before selection,while the lower traces show the convergence of the pool (sequencing fromall colonies surviving a selection on chloramphenicol plates). B. Thesequences of individual clones isolated from each selection and theenhancement in ribosome decoding of UAGA or AGGA codons with cognatetRNAs in O-cat (xxxx103,xxxx146)/tRNAser2(yyyy). C. The enhancement indecoding UAGA codons in the O-cat (UAGA103,UAGA146) gene with ribo-X isdependent on tRNAser2(UCUA).

FIG. 4. The effect of ribo-X on decoding quadruplet codons using severalextended anticodon tRNAs derived from tRNAser2. For the progenitorO-ribosome and ribo-X the chloramphenicol resistance in mg ml-1 withO-cat (XXXX103, XXXX146)/tRNAser2(YYYY) is shown, along with the foldenhancement conferred by ribo-X. The data for UAGG, UAGC, UAGU and UAGcodons are for a reporter with a single selector codon at position 103in O-cat, because the activity was too low to measure accurately in thetwo codon construct.

FIG. 5. The efficiency of ribo-X in decoding the UAGA quadruplet codon.Thin-layer chromatography (TLC) showing the acetylation ofchloramphenicol (Cm) to acetylated chloramphenicol (AcCm) bychloramphenicol acetyl transferase produced from either O-cat or O-cat(UAGA103, UAGA146).

FIG. 6. The specificity of ribo-X. A. Chloramphenicol resistance ofcells containing O-cat (UAGN103, UAGN146)/tRNAser2(UCUA) and ribo-X. B.TLC labelled as in FIG. 5, using constructs as in FIG. 6 panel A.

FIG. 7. Diverging the decoding properties of natural and orthogonalribosomes. The natural ribosome (grey) and the progenitor orthogonalribosome (green) decode wt-(black) and orthogonal-(purple) mRNAsrespectively. Because RF-1 (blue) competes efficiently (dark greyarrows) for UAG codons in the A-site of both ribosomes, amber suppressortRNAs (yellow), that may be uniquely aminoacylated with an unnaturalamino acid, are decoded with equal and low efficiency (light greyarrows) on both ribosomes. Synthetic evolution of the orthogonalribosome leads to an evolved scenario in which a mutant (orange patch)orthogonal ribosome more efficiently decodes amber suppressor tRNAswithin the context of orthogonal mRNAs. Decoding of natural mRNAs isunaffected because the orthogonal ribosome does not read natural mRNAsand the natural ribosome is unaltered. Surface structure figures arecreated using Pymol v0.99 and PDB IDs 2B64 and 1J1U.

FIG. 8. Design of ribosome decoding libraries. A. Structure of a tRNAanticodon stem loop (yellow) bound to mRNA (purple) in the A site of theribosome (green). The 530 loop is shown in orange. B. Structural modelof RF-1 (blue) bound in the A-site of the ribosome C. Secondarystructure of the 530 loop. The boxed sequences form a pseudo knot, ψ ispseudouridine and m⁷G is 7-methyl guanosine. D. The sequence of ribosomedecoding libraries. 3D structure figures were created using Pymol v0.99and PDB IDs 1IBM and 2B64.

FIG. 9. Selection and phenotypic characterization of ribo-X. A. Theselection of ribosomes that decode UAGA and UAG codons, using cognatetRNAs derived from tRNA^(ser2). The top sequence trace shows the 16SrDNA before selection, while the lower traces show the convergence ofthe pool (sequencing from all colonies surviving a selection onchloramphenicol plates). B. The ribo-X, tRNA^(ser2) (UCUA) dependentenhancement in decoding UAGA codons in the O-cat (UAGA103, UAGA146) genemeasured by survival on chloramphenicol. C. As in B, but measuring CATactivity directly. Thin-layer chromatography (TLC) showing theacetylation of chloramphenicol (Cm) to acetylated chloramphenicol (AcCm)by CAT produced from either O-cat or O-cat (UAGA103, UAGA146).

FIG. 10. The translational fidelity of ribo-X is comparable to that ofthe natural ribosome. A. Translation from O-gst-malE is dependent onribo-X or the O-ribosome. B. Ribo-X synthesizes proteins of identicalcomposition to those synthesized by the wild-type ribosome, as judged byelectrospray ionization mass spectrometry. The electrospray ionisationspectra of MBP synthesized by ribo-X, the progenitor O-ribosome or thewild-type ribosome is shown. Each ribosome was used to synthesize theGST-MBP protein, which was purified on glutathione sepharose and subjectto thrombin cleavage at a site in the linker (FIG. 16). The resultingpairs of fragments have identical electrospray ionization spectra(Found: O-ribosome 44984 Da, ribo-X 44984 Da, wt ribosome 44984 Da,expected 44981 Da). C. The translational error frequency measured by³⁵S-cysteine mis-incorporation is indistinguishable for ribo-X and thenatural ribosome. GST-MBP was synthesized by each ribosome in thepresence of ³⁵S-cysteine, purified on glutathione sepharose and digestedwith thrombin. The left panel shows a coomassie stain of the thrombindigest. The un-annotated bands result primarily from the thrombinpreparation. The right panel shows ³⁵S labelling of proteins in asimilar gel, imaged using a Storm Phosphoimager, a coomassie stain ofthe ³⁵S gel is shown in FIG. 19. Lanes 1-3 show thrombin cleavagereactions of purified protein derived from cells containingpSC101*-ribo-X & pO-gst-malE, pSC101*-O-ribosome and pO-gst-malE, andpSC101*-BD and pgst-malE. Lane 4 is a negative control in which cellslacking a gst-malE gene fusion were treated identically to the othersamples. The size markers are pre-stained standards (Bio-Rad 161-0305)D. The translational fidelity of ribo-X is comparable to that of thenatural ribosome as measured by a dual luciferase assay. In this systema C-terminal firefly luciferase is mutated at codon K529(AAA), whichcodes for an essential lysine residue. The extent to which the mutantcodon is misread by tRNA^(Lys)(UUU) is determined by comparing thefirefly luciferase activity resulting from the expression of the mutantgene to the wild-type firefly luciferase, and normalizing anyvariability in expression using the activity of the co-translatedN-terminal Renilla luciferase. Previous work has demonstrated thatmeasured firefly luciferase activities in this system result primarilyfrom the synthesis of a small amount of protein that mis-incorporateslysine in response to the mutant codon, rather than a low activityresulting from the more abundant protein containing encoded mutations³⁷.In experiments examining the fidelity of ribo-X, lysate from cellscontaining pSC101*-ribo-X and pO-DLR and its codon 529 variants wereassayed. Control experiments used lysates from cells containingpSC101*-O-ribosome and pO-DLR and its codon 529 variants or pwt-DLR andits variants. Assays on pO-DLR in the presence and absence of theorthogonal ribosome or ribo-X indicate that greater than 98% oftranslation on pO-DLR is derived from ribo-X or the orthogonal ribosome(FIG. 17), confirming that the fidelity measurements on pO-DLR reflectthe activity of ribo-X.

FIG. 11. A. Ribo-X enhances the efficiency of BpaRS/tRNA_(CUA) dependentunnatural amino acid incorporation in response to single and double UAGcodons. In each lane an equal volume of protein purified fromglutathione sepharose under identical conditions is loaded. Ribo-X isproduced from pSC101*-ribo-X derived rRNA. Bpa isp-benzoyl-L-phenylalanine. BpaRS is p-benzoyl-L-phenylalanyl-tRNAsynthetase. BpaRS/tRNA_(CUA) are produced from pSUPBpa²³ that containssix copies of MjtRNA_(CUA) and is the most efficient unnatural aminoincorporation vector reported to date. (UAG)_(n) describes the number ofstop codons (n) between gst and malE in O-gst(UAG)_(n)malE orgst(UAG)_(n)malE. Lane 10 is from a different gel. The markers are asdescribed in FIG. 9. B. The mass of protein expressed fromO-gst(UAG)₂malE by ribo-X is as expected for the incorporation of 2Bpas. Purified full-length protein was cleaved with thrombin to producean MBP fragment amenable to accurate mass determination. The found mass(45191) is identical to the expected mass for incorporation of two Bpasinto MBP (45191.6). The small peak at 45216 Da is the Na⁺ adduct. C & D.MS/MS fragmentation of chymotryptic peptides derived from GST-MBPsynthesized by ribo-X and incorporating 2 Bpas. The spectra confirm Bpaincorporation at both the expected sites. The fragmentation sites foreach fragment ion are illustrated above the spectra. B denotes Bpa.

FIG. 12 shows supplementary table 1.

FIG. 13 shows supplementary table 2.

FIG. 14 shows the anticodon stem loops of the tRNAs used.

FIG. 15 shows the context of UAGA and UAG selector codons in catreporter genes.

FIG. 16 shows the linker region of the gst-malE expression construct.The codons mutated in gst(UAG)_(n)malE constructs are indicated. Thethrombin cleavage site in GST-MBP is indicated.

FIG. 17 shows the ribosome dependence of O-DLR derived renillaluciferase (O-R-luc) activity. O-ribosomes or ribo-X lead to a 40-45fold activation, indicating that in the presence of O-ribosomes greaterthan 97% of the luciferase fusion is produced by O-ribosomes. The errorbars indicate the standard error.

FIG. 18 shows the Ribo-X mediated enhanced unnatural amino acidincorporation efficiency is robust in minimal medium. Experiments wereperformed as described for FIG. 11A, except that minimal medium was usedfor expression. Similar results were observed when the efficiency of theprogenitor ribosome was compared to Ribo-X. Molecular weight markers areas described in FIG. 10.

FIG. 19 shows 35S misincorporation in GST-MBP. The coomassie gel fromwhich the 35S data in FIG. 10 was acquired and the corresponding 35Simage are shown side-by-side. The alignment of the bands is indicated bythe bounding boxes. The lanes are arranged identically to those in FIG.10 c.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

As the term “orthogonal” is used herein, it refers to a nucleic acid,for example rRNA or mRNA, which differs from natural, endogenous nucleicacid in its ability to cooperate with other nucleic acids. OrthogonalmRNA, rRNA and tRNA are provided in matched groups (cognate groups)which cooperate efficiently. For example, orthogonal rRNA, when part ofa ribosome, will efficiently translate matched cognate orthogonal mRNA,but not natural, endogenous mRNA. For simplicity, a ribosome comprisingan orthogonal rRNA is referred to herein as an “orthogonal ribosome,”and an orthogonal ribosome will efficiently translate a cognateorthogonal mRNA.

An orthogonal codon or orthogonal mRNA codon is a codon in orthogonalmRNA which is only translated by a cognate orthogonal ribosome, ortranslated more efficiently, or differently, by a cognate orthogonalribosome than by a natural, endogenous ribosome. Orthogonal isabbreviated to O (as in O-mRNA).

Thus, by way of example, orthogonal ribosome (O-ribosome)•orthogonalmRNA (O-mRNA) pairs are composed of: an mRNA containing a ribosomebinding site that does not direct translation by the endogenousribosome, and an orthogonal ribosome that efficiently and specificallytranslates the orthogonal mRNA, but does not appreciably translatecellular mRNAs.

“Evolved”, as applied herein for example in the expression “evolvedorthogonal ribosome”, refers to the development of a function of amolecule through diversification and selection. For example, a libraryof rRNA molecules diversified at desired positions can be subjected toselection according to the procedures described herein. An evolved rRNAis obtained by the selection process.

As used herein, the term “mRNA” when used in the context of an O-mRNAO-ribosome pair refers to an mRNA that comprises an orthogonal codonwhich is efficiently translated by a cognate O-ribosome, but not by anatural, wild-type ribosome. In addition, it may comprise an mutantribosome binding site (particularly the sequence from the AUG initiationcodon upstream to −13 relative to the AUG) that efficiently mediates theinitiation of translation by the O-ribosome, but not by a wild-typeribosome. The remainder of the mRNA can vary, such that placing thecoding sequence for any protein downstream of that ribosome binding sitewill result in an mRNA that is translated efficiently by the orthogonalribosome, but not by an endogenous ribosome.

As used herein, the term “rRNA” when used in the context of an O-mRNAO-ribosome pair refers to a rRNA mutated such that the rRNA is anorthogonal rRNA, and a ribosome containing it is an orthogonal ribosome,i.e., it efficiently translates only a cognate orthogonal mRNA. Theprimary, secondary and tertiary structures of wild-type ribosomal rRNAsare very well known, as are the functions of the various conservedstructures (stems-loops, hairpins, hinges, etc.). O-rRNA typicallycomprises a mutation in 16S rRNA which is responsible for binding oftRNA during the translation process. It may also comprise mutations inthe 3′ regions of the small rRNA subunit which are responsible for theinitiation of translation and interaction with the ribosome binding siteof mRNA.

The expression of an “O-rRNA” in a cell, as the term is used herein, isnot toxic to the cell. Toxicity is measured by cell death, oralternatively, by a slowing in the growth rate by 80% or more relativeto a cell that does not express the “O-mRNA.” Expression of an O-rRNAwill preferably slow growth by less than 50%, preferably less than 25%,more preferably less than 10%, and more preferably still, not at all,relative to the growth of similar cells lacking the O-rRNA.

As used herein, the terms “more efficiently translates” and “moreefficiently mediates translation” mean that a given O-mRNA is translatedby a cognate O-ribosome at least 25% more efficiently, and preferably atleast 2, 3, 4 or 8 or more times as efficiently as an O-mRNA istranslated by a wild-type ribosome or a non-cognate O-ribosome in thesame cell or cell type. As a gauge, for example, one may evaluatetranslation efficiency relative to the translation of an O-mRNA encodingchloramphenicol acetyl transferase using at least one orthogonal codonby a natural or non-cognate orthogonal ribosome.

As used herein, the term “corresponding to” when used in reference tonucleotide sequence means that a given sequence in one molecule, e.g.,in a 16S rRNA, is in the same position in another molecule, e.g., a 16SrRNA from another species. By “in the same position” is meant that the“corresponding” sequences are aligned with each other when aligned usingthe BLAST sequence alignment algorithm “BLAST 2 Sequences” described byTatusova and Madden (1999, “Blast 2 sequences—a new tool for comparingprotein and nucleotide sequences”, FEMS Microbiol. Lett. 174:247-250)and available from the U.S. National Center for BiotechnologyInformation (NCBI). To avoid any doubt, the BLAST version 2.2.11(available for use on the NCBI website or, alternatively, available fordownload from that site) is used, with default parameters as follows:program, blastn; reward for a match, 1; penalty for a mismatch, −2; opengap and extend gap penalties 5 and 2, respectively; ga×dropoff, 50;expect 10.0; word size 11; and filter on.

As used herein, the term “selectable marker” refers to a gene sequencethat permits selection for cells in a population that encode and expressthat gene sequence by the addition of a corresponding selection agent.

As used herein, the term “region comprising sequence that interacts withmRNA at the ribosome binding site” refers to a region of sequencecomprising the nucleotides near the 3′ terminus of 16S rRNA thatphysically interact, e.g., by base pairing or other interaction, withmRNA during the initiation of translation. The “region” includesnucleotides that base pair or otherwise physically interact withnucleotides in mRNA at the ribosome binding site, and nucleotides withinfive nucleotides 5′ or 3′ of such nucleotides. Also included in this“region” are bases corresponding to nucleotides 722 and 723 of the E.coli 16S rRNA, which form a bulge proximal to the minor groove of theShine-Delgarno helix formed between the ribosome and mRNA.

As used herein, the term “diversified” means that individual members ofa library will vary in sequence at a given site. Methods of introducingdiversity are well known to those skilled in the art, and can introducerandom or less than fully random diversity at a given site. By “fullyrandom” is meant that a given nucleotide can be any of G, A, T, or C (orin RNA, any of G, A, U and C). By “less than fully random” is meant thata given site can be occupied by more than one different nucleotide, butnot all of G, A, T (U in RNA) or C, for example where diversity permitseither G or A, but not U or C, or permits G, A, or U but not C at agiven site.

As used herein, the term “ribosome binding site” refers to the region ofan mRNA that is bound by the ribosome at the initiation of translation.As defined herein, the “ribosome binding site” of prokaryotic mRNAsincludes the Shine-Delgarno consensus sequence and nucleotides −13 to +1relative to the AUG initiation codon.

As used herein, the term “unnatural amino acid” refers to an amino acidother than the amino acids that occur naturally in protein. Non-limitingexamples include: a p-acetyl-L-phenylalanine, a p-iodo-L-phenylalanine,an O-methyl-L-tyrosine, a p-propargyloxyphenylalanine, ap-propargyl-phenylalanine, an L-3-(2-naphthyl)alanine, a3-methyl-phenylalanine, an O-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine,a tri-O-acetyl-GlcNAcpβ-serine, an L-Dopa, a fluorinated phenylalanine,an isopropyl-L-phenylalanine, a p-azido-L-phenylalanine, ap-acyl-L-phenylalanine, a p-benzoyl-L-phenylalanine, an L-phosphoserine,a phosphonoserine, a phosphonotyrosine, a p-bromophenylalanine, ap-amino-L-phenylalanine, an isopropyl-L-phenylalanine, an unnaturalanalogue of a tyrosine amino acid; an unnatural analogue of a glutamineamino acid; an unnatural analogue of a phenylalanine amino acid; anunnatural analogue of a serine amino acid; an unnatural analogue of athreonine amino acid; an alkyl, aryl, acyl, azido, cyano, halo,hydrazine, hydrazide, hydroxyl, alkenyl, alkynl, ether, thiol, sulfonyl,seleno, ester, thioacid, borate, boronate, phospho, phosphono,phosphine, heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, oramino substituted amino acid, or a combination thereof; an amino acidwith a photoactivatable cross-linker; a spin-labeled amino acid; afluorescent amino acid; a metal binding amino acid; a metal-containingamino acid; a radioactive amino acid; a photocaged and/orphotoisomerizable amino acid; a biotin or biotin-analogue containingamino acid; a keto containing amino acid; an amino acid comprisingpolyethylene glycol or polyether; a heavy atom substituted amino acid; achemically cleavable or photocleavable amino acid; an amino acid with anelongated side chain; an amino acid containing a toxic group; a sugarsubstituted amino acid; a carbon-linked sugar-containing amino acid; aredox-active amino acid; an α-hydroxy containing acid; an amino thioacid; an α,α disubstituted amino acid; a β-amino acid; a cyclic aminoacid other than proline or histidine, and an aromatic amino acid otherthan phenylalanine, tyrosine or tryptophan.

In our copending international patent application PCT/GB2006/002637 wedescribe the generation of orthogonal ribosome/mRNA pairs in which theribosome binding site in the O-mRNA binds specifically to theO-ribosome.

Briefly, the bacterial ribosome is a 2.5 MDa complex of rRNA and proteinresponsible for translation of mRNA into protein (The Ribosome, Vol.LXVI. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.;2001). The interaction between the mRNA and the 30S subunit of theribosome is an early event in translation (Laursen, B. S., Sorensen, H.P., Mortensen, K. K. & Sperling-Petersen, H. U., Microbiol Mol Biol Rev69, 101-123 (2005)), and several features of the mRNA are known tocontrol the expression of a gene, including the first codon (Wikstrom,P. M., Lind, L. K., Berg, D. E. & Bjork, G. R., J Mol Biol 224, 949-966(1992)), the ribosome-binding sequence (including the Shine Delgarno(SD) sequence (Shine, J. & Delgarno, L., Biochem J 141, 609-615 (1974),Steitz, J. A. & Jakes, K., Proc Natl Acad Sci USA 72, 4734-4738 (1975),Yusupova, G. Z., Yusupov, M. M., Cate, J. H. & Noller, H. F., Cell 106,233-241 (2001)), and the spacing between these sequences (Chen, H.,Bjerknes, M., Kumar, R. & Jay, E., Nucleic Acids Res 22, 4953-4957(1994)). In certain cases mRNA structure (Gottesman, S. et al. in TheRibosome, Vol. LXVI (Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.; 2001), Looman, A. C., Bodlaender, J., de Gruyter, M.,Vogelaar, A. & van Knippenberg, P. H., Nucleic Acids Res 14, 5481-5497(1986)), Liebhaber, S. A., Cash, F. & Eshleman, S. S., J Mol Biol 226,609-621 (1992), or metabolite binding (Winkler, W., Nahvi, A. & Breaker,R. R., Nature 419, 952-956 (2002)), influences translation initiation,and in rare cases mRNAs can be translated without a SD sequence, thoughtranslation of these sequences is inefficient (Laursen, B. S., Sorensen,H. P., Mortensen, K. K. & Sperling-Petersen, H. U., Microbiol Mol BiolRev 69, 101-123 (2005)), and operates through an alternate initiationpathway, Laursen, B. S., Sorensen, H. P., Mortensen, K. K. &Sperling-Petersen, H. U. Initiation of protein synthesis in bacteria.Microbiol Mol Biol Rev 69, 101-123 (2005). For the vast majority ofbacterial genes the SD region of the mRNA is a major determinant oftranslational efficiency. The classic SD sequence GGAGG interactsthrough RNA-RNA base-pairing with a region at the 3′ end of the 16S rRNAcontaining the sequence CCUCC, known as the Anti Shine Delgarno (ASD).In E. coli there are an estimated 4,122 translational starts(Shultzaberger, R. K., Bucheimer, R. E., Rudd, K. E. & Schneider, T. D.,J Mol Biol 313, 215-228 (2001)), and these differ in the spacing betweenthe SD-like sequence and the AUG start codon, the degree ofcomplementarity between the SD-like sequence and the ribosome, and theexact region of sequence at the 3′ end of the 16S rRNA with which themRNA interacts. The ribosome therefore drives translation from a morecomplex set of sequences than just the classic Shine Delgarno (SD)sequence. For clarity, mRNA sequences believed to bind the 3′ end of 16SrRNA are referred to as SD sequences and to the specific sequence GGAGGis referred to as the classic SD sequence.

Mutations in the SD sequence often lead to rapid cell lysis and death(Lee, K., Holland-Staley, C. A. & Cunningham, P. R., RNA 2, 1270-1285(1996), Wood, T. K. & Peretti, S. W., Biotechnol. Bioeng 38, 891-906(1991)). Such mutant ribosomes mis-regulate cellular translation and arenot orthogonal. The sensitivity of cell survival to mutations in the ASDregion is underscored by the observation that even a single change inthe ASD can lead to cell death through catastrophic and globalmis-regulation of proteome synthesis (Jacob, W. F., Santer, M. &Dahlberg, A. E., Proc Natl Acad Sci USA 84, 4757-4761 (1987). Othermutations in the rRNA can lead to inadequacies in processing or assemblyof functional ribosomes.

PCT/GB2006/02637 describes methods for tailoring the molecularspecificity of duplicated E. coli ribosome mRNA pairs with respect tothe wild-type ribosome and mRNAs to produce multiple orthogonal ribosomeorthogonal mRNA pairs. In these pairs the ribosome efficientlytranslates only the orthogonal mRNA and the orthogonal mRNA is not anefficient substrate for cellular ribosomes. Orthogonal ribosomes asdescribed therein that do not translate endogenous mRNAs permit specifictranslation of desired cognate mRNAs without interfering with cellulargene expression. The network of interactions between these orthogonalpairs is predicted and measured, and it is shown that orthogonalribosome mRNA pairs can be used to post-transcriptionally program thecell with Boolean logic.

PCT/GB2006/02637 describes a mechanism for positive and negativeselection for evolution of orthogonal translational machinery. Theselection methods are applied to evolving multiple orthogonal ribosomemRNA pairs (O-ribosome O-mRNA). Also described is the successfulprediction of the network of interactions between cognate andnon-cognate O-ribosomes and O-mRNAs.

Here we provide new, further modified orthogonal ribosomes and methodsfor producing such O-ribosomes which expand the molecular decodingproperties of the ribosome. Specifically, we evolve orthogonal ribosomesthat more efficiently decode a set of quadruplet codons using tRNAs withextended anticodon loops. Moreover, we provide a mechanistic explanationof the enhancements we observe, and demonstrate that the evolvedorthogonal ribosome is also substantially more efficient at decodingamber codons with amber suppressor tRNAs.

We disclose evolved orthogonal ribosomes which enhance the efficiency ofsynthetic genetic code expansion. We provide cellular modules composedof an orthogonal ribosome and an orthogonal mRNA. These pairs functionin parallel with, but independent of, the natural ribosome-mRNA pair inEscherichia coli. Orthogonal ribosomes do not synthesize the proteomeand may be diverged to operate using different tRNA decoding rules fromnatural ribosomes. Here we demonstrate the evolution of an orthogonalribosome (ribo-X) for the efficient, high fidelity decoding of codonssuch as amber codons placed within the context of an orthogonal mRNA inliving cells. We combine ribo-X, orthogonal mRNAs and orthogonalaminoacyl-tRNA synthetase/tRNA pairs to substantially increase theefficiency of site-specific unnatural amino acid incorporation in E.coli. This advantageously allows the efficient synthesis of proteinsincorporating unnatural amino acids at multiple sites, and/or minimizesthe functional and/or phenotypic effects of truncated proteins forexample in experiments that use unnatural amino acid incorporation toprobe protein function in vivo.

Orthogonal Codons

For the first time, we describe an evolved ribosome which is capable oftranslating an orthogonal mRNA codon, which means that the ribosomeinterprets mRNA information according to a code which is not theuniversal genetic code, but an orthogonal genetic code. This introducesa number of possibilities, including the possibility of having twoseparate genetic systems present in the cell, wherein cross-talk iseliminated by virtue of the difference in code; or of a mRNA moleculeencoding different polypeptides according to which code is used totranslate it.

An orthogonal codon, from which orthogonal genetic codes can beassembled, is a code which is other than the universal triplet code.Table 1 below represents the universal genetic code:

TABLE 1 Second nucleotide U C A G U UUU UCU UAU UGU U Phenyl- SerineTyrosine Cysteine alanine (Ser) (Tyr) (Cys) (Phe) UUC Phe UCC Ser UACTyr UGC Cys C UUA UCA Ser UAA STOP UGA STOP A Leucine (Leu) UUG Leu UCGSer UAG STOP UGG G Tryptophan (Trp) C CUU CCU CAU CGU U Leucine ProlineHistidine Arginine (Leu) (Pro) (His) (Arg) CUC Leu CCC Pro CAC His CGCArg C CUA Leu CCA Pro CAA CGA Arg A Glutamine (Gln) CUG Leu CCG Pro CAGGln CCG Arg G A AUU ACU AAU AGU U Isoleucine Threonine Asparagine Serine(Ile) (Thr) (Asn) (Ser) AUC Ile ACC Thr AAC Asn AGC Ser C AUA Ile ACAThr AAA Lysine AGA A (Lys) Arginine (Arg) AUG ACG Thr AAG Lys AGG Arg GMethio- nine (Met) or START G GUU GCU GAU GGU U Valine Alanine AsparticGlycine Val (Ala) acid (Gly) (Asp) GUC (Val) GCC Ala GAC Asp GGC Gly CGUA Val GCA Ala GAA GGA Gly A Glutamic acid (Glu) GUG Val GCG Ala GAGGlu GGG Gly G

Certain variations in this code occur naturally; for example,mitochondria use UGA to encode tryptophan (Trp) rather than as a chainterminator. In addition,

-   -   most animal mitochondria use AUA for methionine not isoleucine        and    -   all vertebrate mitochondria use AGA and AGG as chain        terminators.    -   Yeast mitochondria assign all codons beginning with CU to        threonine instead of leucine (which is still encoded by UUA and        UUG as it is in cytosolic mRNA).

Plant mitochondria use the universal code, and this has permittedangiosperms to transfer mitochondrial genes to their nucleus with greatease.

Violations of the universal code are far rarer for nuclear genes. A fewunicellular eukaryotes have been found that use one or two (of theirthree) STOP codons for amino acids instead.

The vast majority of proteins are assembled from the 20 amino acidslisted above even though some of these may be chemically altered, e.g.by phosphorylation, at a later time.

However, two cases have been found in nature where an amino acid that isnot one of the standard 20 is inserted by a tRNA into the growingpolypeptide.

-   -   Selenocysteine. This amino acid is encoded by UGA. UGA is still        used as a chain terminator, but the translation machinery is        able to discriminate when a UGA codon should be used for        selenocysteine rather than STOP. This codon usage has been found        in certain Archaea, eubacteria, and animals (humans synthesize        25 different proteins containing selenium).    -   Pyrrolysine. In one gene found in a member of the Archaea, this        amino acid is encoded by UAG. How the translation machinery        knows when it encounters UAG whether to insert a tRNA with        pyrrolysine or to stop translation is not yet known.

All of the above are, for the purposes of the present invention,considered to be part of the universal genetic code.

The present invention enables novel codes, not previously known innature, to be developed and used in the context of orthogonal mRNA/rRNApairs.

Selection for Orthogonal Ribosomes

A selection approach for the identification of orthogonal ribosomeorthogonal mRNA pairs, or other pairs of orthogonal molecules, requiresselection for translation of orthogonal codons in O-mRNA. The selectionis advantageously positive selection, such that cells which expressO-mRNA are selected over those that do not, or do so less efficiently.

A number of different positive selection agents can be used. The mostcommon selection strategies involve conditional survival on antibiotics.Of these positive selections, the chloramphenicol acetyl-transferasegene in combination with the antibiotic chloramphenicol has proved oneof the most useful. Others as known in the art, such as ampicillin,kanamycin, tetracycline or streptomycin resistance, among others, canalso be used.

O-mRNA/O-rRNA pairs can be used to produce an orthogonal transcript in ahost cell, for example CAT, that can only be translated by the cognateorthogonal ribosome, thereby permitting extremely sensitive control ofthe expression of a polypeptide encoded by the transcript. The pairs canthus be used to produce a polypeptide of interest by, for example,introducing nucleic acid encoding such a pair to a cell, where theorthogonal mRNA encodes the polypeptide of interest. The translation ofthe orthogonal mRNA by the orthogonal ribosome results in production ofthe polypeptide of interest. It is contemplated that polypeptidesproduced in cells encoding orthogonal mRNA orthogonal ribosome pairs caninclude unnatural amino acids.

The methods described herein are applicable to the selection oforthogonal mRNA orthogonal rRNA pairs in species in which the O-mRNAcomprises orthogonal codons which are translated by the O-rRNA. Thus,the methods are broadly applicable across prokaryotic and eukaryoticspecies, in which this mechanism is conserved. The sequence of 16S rRNAis known for a large number of bacterial species and has itself beenused to generate phylogenetic trees defining the evolutionaryrelationships between the bacterial species (reviewed, for example, byLudwig & Schleifer, 1994, FEMS Microbiol. Rev. 15: 155-73; see alsoBergey's Manual of Systematic Bacteriology Volumes 1 and 2, Springer,George M. Garrity, ed.). The Ribosomal Database Project II (Cole J R,Chai B, Farris R J, Wang Q, Kulam S A, McGarrell D M, Garrity G M,Tiedje J M, Nucleic Acids Res, (2005) 33(Database Issue):D294-D296. doi:10.1093/nar/gki038) provides, in release 9.28 (Jun. 17, 2005), 155,708aligned and annotated 16S rRNA sequences, along with online analysistools.

Phylogenetic trees are constructed using, for example, 16S rRNAsequences and the neighbour joining method in the ClustalW sequencealignment algorithm. Using a phylogenetic tree, one can approximate thelikelihood that a given set of mutations (on 16S rRNA and a codon inmRNA) that render the set orthogonal with respect to each other in onespecies will have a similar effect in another species. Thus, themutations rendering mRNA/16S rRNA pairs orthogonal with respect to eachother in one member of, for example, the Enterobacteriaceae Family(e.g., E. coli) would be more likely to result in orthogonalmRNA/orthogonal ribosome pairs in another member of the same Family(e.g., Salmonella) than in a member of a different Family on thephylogenetic tree.

In some instances, where bacterial species are very closely related, itmay be possible to introduce corresponding 16S rRNA and mRNA mutationsthat result in orthogonal molecules in one species into the closelyrelated species to generate an orthogonal mRNA orthogonal rRNA pair inthe related species. Also where bacterial species very are closelyrelated (e.g., for E. coli and Salmonella species), it may be possibleto introduce orthogonal 16S rRNA and orthogonal mRNA from one speciesdirectly to the closely related species to obtain a functionalorthogonal mRNA orthogonal ribosome pair in the related species.

Alternatively, where the species in which one wishes to identifyorthogonal mRNA orthogonal ribosome pairs is not closely related (e.g.,where they are not in the same phylogenetic Family) to a species inwhich a set of pairs has already been selected, one can use selectionmethods as described herein to generate orthogonal mRNA orthogonalribosome pairs in the desired species. Briefly, one can prepare alibrary of mutated orthogonal 16S rRNA molecules. The library can thenbe introduced to the chosen species. One or more O-mRNA sequences can begenerated which comprise a sequence encoding a selection polypeptide asdescribed herein using one or more orthogonal codons (the bacterialspecies must be sensitive to the activity of the selection agents, amatter easily determined by one of skill in the art). The O-mRNA librarycan then be introduced to cells comprising the O-rRNA library, followedby positive selection for those cells expressing the positive selectablemarker in order to identify orthogonal ribosomes that pair with theO-mRNA.

The methods described herein are applicable to the identification ofmolecules useful to direct translation or other processes in a widerange of bacteria, including bacteria of industrial and agriculturalimportance as well as pathogenic bacteria. Pathogenic bacteria are wellknown to those of skill in the art, and sequence information, includingnot only 16S rRNA sequence, but also numerous mRNA coding sequences, areavailable in public databases, such as GenBank. Common, but non-limitingexamples include, e.g., Salmonella species, Clostridium species, e.g.,Clostridium botulinum and Clostridium perfringens, Staphylococcus sp.,e.g, Staphylococcus aureus; Campylobacter species, e.g., Campylobacterjejuni, Yersinia species, e.g., Yersinia pestis, Yersinia enterocoliticaand Yersinia pseudotuberculosis, Listeria species, e.g., Listeriamonocytogenes, Vibrio species, e.g., Vibrio cholerae, Vibrioparahaemolyticus and Vibrio vulnificus, Bacillus cereus, Aeromonasspecies, e.g., Aeromonas hydrophila, Shigella species, Streptococcusspecies, e.g., Streptococcus pyogenes, Streptococcus faecalis,Streptococcus faecium, Streptococcus pneumoniae, Streptococcus durans,and Streptococcus avium, Mycobacterium tuberculosis, Klebsiella species,Enterobacter species, Proteus species, Citrobacter species, Aerobacterspecies, Providencia species, Neisseria species, e.g., Neisseriagonorrhea and Neisseria meningitidis, Heamophilus species, e.g.,Haemophilus influenzae, Helicobacter species, e.g., Helicobacter pylori,Bordetella species, e.g., Bordetella pertussis, Serratia species, andpathogenic species of E. coli, e.g., Enterotoxigenic E. coli (ETEC),enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli O157:H7(EHEC).

Release Factor 1/Amber Codons

Advantageously, to maximize the efficiency of full-length proteinsynthesis with respect to truncated protein, the effects of releasefactor 1 (RF-1)-mediated chain termination would be minimized for theexpression of a gene of interest, while the decoding of chromosomalamber stop codons would remain unaltered. We conceived to use recentlydescribed orthogonal ribosome-mRNA pairs to address this challenge (seeexamples and FIG. 7).

Unlike the natural ribosome the orthogonal ribosome is not responsiblefor synthesizing the proteome, and is therefore tolerant to mutations inthe highly conserved rRNA that cause lethal or dominant negative effectsin the natural ribosome. Orthogonal ribosomes may therefore beadvantageously evolved towards decreased RF-1 binding and increased tRNAdependent amber suppression according to the present invention.Moreover, increased amber suppression within the context of anorthogonal ribosome has the advantage of operating on amber codonswithin orthogonal mRNAs whilst advantageously also not increasingsuppression of chromosomal stop codons.

We disclose the synthetic evolution of an orthogonal ribosome (ribo-X)for the efficient, high fidelity, suppressor tRNA dependent decoding ofamber stop codons placed within the context of an orthogonal mRNA inliving cells. Ribo-X may preferably be combined with orthogonal mRNAsand orthogonal aminoacyl-tRNA synthetase/tRNA_(CUA) pairs toadvantageously significantly increase the efficiency of site-specificunnatural amino acid incorporation in E. coli. This increase inefficiency makes it possible to synthesize proteins incorporatingunnatural amino acids at multiple sites, and minimizes the functionaland phenotypic effects of truncated proteins in vivo. This has clearindustrial application and utility, for example in the manufacture ofproteins incorporating unnatural amino acids.

Since ribo-X increases suppression of amber stop codons by suppressortRNAs of distinct sequence and structure, we suggest that ribo-Xoperates by decreasing its functional interaction with RF-1, allowingthe suppressor tRNAs to more efficiently compete for A-site binding inthe presence of a UAG codon on the mRNA. Variations or optimisation ofthe strategy disclosed here may yield further increases in ambersuppression while maintaining translational fidelity. In this regard itis encouraging both that biochemical evidence suggests distinctconformations of the decoding centre are recognized by tRNAs and RF-1(Youngman et al Cold Spring Harb Symp Quant Biol 71, 545-549 (2006)) andthat we have been able to select combinations of mutations for whichRF-1 mediated termination is decreased without decreasing the fidelityof tRNA decoding. These observations surprisingly indicate that themolecular determinants for the fidelity of tRNA decoding and RF-1binding need not be tightly coupled and thus further independentmodulation within the orthogonal system is enabled by the presentinvention.

By improving amber suppression efficiency on the orthogonal ribosome itis now possible to diverge the decoding properties of the orthogonalribosome from those of the cellular ribosome such that the sameinsertion signal is read with a different efficiency on cellular andorthogonal mRNAs within the same cell as demonstrated herein. Aconceptually similar, but mechanistically distinct, strategy involvinglocalization of specialized translational components is used by natureto direct the incorporation of selenocysteine in response to a subset ofUGA codons. Thus the invention may find application in similarstrategies to enhance the efficiency of synthetic eukaryotic geneticcode expansion. Since the meaning of codons on any mRNA are set by thetranslational machinery that decodes that mRNA, it may be possible touse our approach to write entirely new genetic codes on orthogonal mRNAsand to undo the “frozen accident” of the existing genetic code. Forexample, it may be possible to use tRNAs that are poor substrates forthe cellular ribosome but are efficiently decoded by evolved orthogonalribosomes to write independent and parallel codes (orthogonal geneticcodes). Orthogonal genetic codes may form a basis for a biological“virtual operating system” that further expands the information storageand genetic encoding capacity of the cell.

Bacterial Transformation

The methods described herein rely upon the introduction of foreign orexogenous nucleic acids into bacteria. Methods for bacterialtransformation with exogenous nucleic acid, and particularly forrendering cells competent to take up exogenous nucleic acid, is wellknown in the art. For example, Gram negative bacteria such as E. coliare rendered transformation competent by treatment with multivalentcationic agents such as calcium chloride or rubidium chloride. Grampositive bacteria can be incubated with degradative enzymes to removethe peptidoglycan layer and thus form protoplasts. When the protoplastsare incubated with DNA and polyethylene glycol, one obtains cell fusionand concomitant DNA uptake. In both of these examples, if the DNA islinear, it tends to be sensitive to nucleases so that transformation ismost efficient when it involves the use of covalently closed circularDNA. Alternatively, nuclease-deficient cells (RecBC⁻ strains) can beused to improve transformation.

Electroporation is also well known for the introduction of nucleic acidto bacterial cells. Methods are well known, for example, forelectroporation of Gram negative bacteria such as E. coli, but are alsowell known for the electroporation of Gram positive bacteria, such asEnterococcus faecalis, among others, as described, e.g., by Dunny etal., 1991, Appl. Environ. Microbiol. 57: 1194-1201.

EXAMPLES Example 1 Evolution of an Orthogonal Ribosome

Design of a Ribosome Decoding Centre Library for Enhanced QuadrupletDecoding

While the detailed mechanism of quadruplet decoding iscontroversial^(17, 25) and may differ for different tRNA/codon pairs, itis clear that an early and essential step in decoding is the binding ofthe extended anticodon tRNA to the A-site of the ribosome in response tothe quadruplet codon. Since the A-site is the gateway to the tRNAtranslocation corridor composed of the A, P and E sites²⁶⁻²⁸, and is theprimary site of codon-anticodon proofreading for elongator tRNAs²⁹, wereasoned that combinations of mutations in 16S rRNA, which forms theA-site, might yield an variant A-site that functions more efficientlywith an extended anticodon loop tRNA.

To design an A-site library we examined the structures of normal tRNAanticodon stem loops bound to the ribosomal A site^(27, 28, 30). Thesestructures show that the 530 loop in 16S rRNA is proximal to, andintimately associated with, the codon-anticodon helix (FIG. 1). Wetherefore randomized the seven nucleotides 529-535 in the 530 loop toall combinations of nucleotides to create an N7 library (FIG. 1).Moreover, we reasoned that since the extended tRNA anticodon contains anadditional nucleotide, shorter 530 loop sequences in 16S rRNA mightprovide more space to accommodate the extended anticodon whereas longer530 loop sequences might provide greater flexibility for the rRNA toadapt to the extended anticodon. We therefore created four additionallibraries (N5, N6, N8, and N9 (FIG. 1)) All libraries created were morethan 99% complete as determined by Poisson sampling statistics.

Selection for Orthogonal Ribosomes with Enhanced Quadruplet Decoding

To create a selection system for ribosomes that more efficiently read aquadruplet codon we required a reporter of orthogonal ribosome activitythat contains selector quadruplet codons. We introduced the quadrupletcodons (either UAGA or AGGA) at two sites in chloramphenicol acetyltransferase (cat) gene (Ser103 and Ser 146, an essential and conservedcatalytic serine residue³¹ that insures the fidelity of incorporation)downstream of an orthogonal ribosome binding site (FIG. 2). We combinedthe resulting reporters with well-characterized UCUA or UCCU anticodontRNAs derived from tRNA^(ser2) (FIG. 2), previously selected by Maglieryet al⁸. Cells containing O-cat (UAGA103, UAGA146)/tRNA^(ser2) (UCUA) andthe O-ribosome had an IC₅₀ on chloramphenicol of 25 mg ml⁻¹, and cellscontaining O-cat (AGGA103, AGGA146)/tRNA^(ser2) (UCCU) had an IC₅₀ onchloramphenicol of 80 mg ml⁻¹. For comparison the wild-type O-catsupports growth to 500 mg ml⁻¹ chloramphenicol in the presence of acognate O-ribosome.

To select mutant ribosomes that more efficiently decode the UAGA codonwe combined each orthogonal ribosome library (N5-N9) with O-cat(UAGA103, UAGA146)/tRNA^(ser2) (UCUA) and challenged the cells to growon chloramphenicol concentrations at which the O-ribosome does notsupport growth (FIG. 2). While no clones containing insertions ordeletions survived for libraries N5, N6, N8 and N9, suggesting that the530 loop is intolerant to longer or shorter sequences of anycomposition, clones did survive from the N7 library on 50 mg ml⁻¹chloramphenicol. We isolated and sequenced ten plasmids encodingsurviving N7 library members. For the UAGA decoding selection, allclones sequenced were identical, and contained the mutations A531G andU534A. (FIG. 3). Next, we repeated the selection using the AGGA reporterand cognate tRNA. In this selection we found that the library alsoconverges, to sequences similar to, and in some cases identical to, thesequence selected for decoding UAGA (FIG. 3). We combined the ribosomesfrom both selections with either the AGGA or UAGA reporters and foundthat, as expected, the UAGA selected ribosome decodes UAGA mostefficiently. We also found that the UAGA selected ribosome is among themost efficient ribosomes for AGGA decoding. We therefore decided tocharacterize the A531G, U534A mutant ribosome, which we refer to asribo-X, in more detail.

Example 2 Analysis of Ribo-X

Ribo-X Enhances tRNA Dependent Reading of UAGA Quadruplet Codons

To investigate whether ribo-X reads quadruplet codons in a tRNAdependent manner, we co-transformed ribo-X and O-cat (UAGA103, UAGA146)and measured chloramphenicol resistance. We find that ribo-X does notsignificantly contribute to +1 frame shifting in the absence of anextended anticodon tRNA (Cm resistance <1 mg ml⁻¹) (FIG. 3).

To measure the extent to which ribo-X enhances tRNA^(ser2) (UCUA)dependent decoding of the UAGA codon we compared the chloramphenicolresistance of cells transformed with O-cat (UAGA103,UAGA146)/tRNA^(ser2) (UCUA) and either ribo-X or the progenitorO-ribosome. We find that the ribo-X containing cells survive onconcentrations of chloramphenicol five times higher (125 mg ml⁻¹) thancells containing the progenitor O-ribosome (25 mg ml⁻¹) (FIG. 3). Theenhanced quadruplet decoding was further confirmed by in vitro CATassays³² (FIG. 5), which also confirm that resistance is linear withchloramphenicol acetyl transferase activity (not shown). To ascertain ifthe enhanced activity of ribo-X on quadruplet codons is at the expenseof triplet decoding we compared the chloramphenicol resistance conferredby ribo-X to that conferred by the progenitor O-ribosome on cellscontaining O-cat. We find no difference in activity; both ribosomessupport growth to 500 mg ml⁻¹, indicating that ribo-X is efficient atreading triplet codons.

Ribo-X Shows Specificity for Fourth Base Interaction & PreferentiallyDecodes Quadruplet over Triplet Codons with Extended Anticodon tRNAs

We investigated the specificity of ribo-X for Watson-Crick base pairingin the fourth position of the codon-anticodon interaction by measuringthe chloramphenicol acetyl transferase activity of ribo-X with O-cat(UAGN103, UAGN146)/tRNA^(ser2) (UCUA). We find that ribo-X shows aselectivity of more than ten-fold for the cognate A:U pair, over anyother base-pair at the fourth position. (FIG. 6) consistent withprevious reports for the natural ribosome³³.

To investigate whether ribo-X preferentially decodes a quadruplet codonover a triplet codon, when supplied with an extended anticodon tRNA, wecompared the chloramphenicol resistance conferred by an O-cat gene inwhich codon 103 is UAG and codon 104 begins with an A (O-frame UAGA,FIG. 2) with that conferred by an O-cat gene containing an in-frame UAGAquadruplet codon at position 103. We find that ribo-X and tRNA^(ser2)(UCUA) confer resistance to chloramphenicol up to 20 mg ml⁻¹ on thetriplet codon reporter and up to 400 mg ml⁻¹ on the quadruplet codonreporter. These data suggest that less than 5% of the termination onquadruplet codons with ribo-X results from triplet decoding, and thatwith the extended anticodon tRNA, ribo-X prefers quadruplet over tripletdecoding by approximately 10-fold.

Ribo-X Shows Increased Efficiency in Decoding Quadruplet Codons

To begin to explore the extent to which enhanced quadruplet decoding ofUAGA and AGGA with ribo-X is portable to other extended anticodons, wefirst altered the anticodon of the tRNA to GCUA, CCUA, or ACUA andaltered the codons at position 103 in the O-cat gene to theirWatson-Crick complement (UAGC, UAGG, UAGU). Ribo-X shows an up to 8-foldincrease in efficiency at decoding quadruplet codons with respect to theprogenitor O-ribosome (FIG. 4). In addition we find that ribo-X enhancestranslational efficiency 2.5-fold with a CCCU reporter in the presenceof a cognate extended anticodon tRNA.

Ribo-X Increases Amber Decoding and UAGN decoding by DecreasingFunctional Interactions with Release Factor.

The decoding enhancement with UAGN codons is generally larger than thatobserved with AGGA and CCCU codons, and this prompted us to ask whetherribo-X enhances decoding of UAGN codons in part by decreasing itsfunctional interaction with release factor 1 (RF1). The ribosome bindsRF1 in response to amber codons in the ribosomal A site, and causespeptide chain termination³⁴. RF1-mediated termination is thereforebelieved to compete with amber suppressor tRNA mediated peptide chainelongation in response to an amber codon in the ribosomal A-site.Ribosomes that do not functionally interact with RF1 but are still ableto perform protein synthesis and function with NCUA tRNAs would be moreefficient than un-evolved ribosomes at decoding UAGN codons. If theenhanced, activity of ribo-X on UAGN codons were due in part to adecreased functional interaction with RF1 then ribo-X should be moreefficient at decoding the amber stop codon (UAG) with an ambersuppressor tRNA. Indeed we find that when we contract the anticodon fromUCUA to CUA, the resulting suppressor tRNA is 8 times more efficient atdecoding an amber codon at position 103 in O-cat with ribo-X than withthe progenitor O-ribosome (FIG. 4). This suggests that a major mechanismby which ribo-X increases its efficiency on UAGN codons is by decreasingits functional interaction with release factor, and provides evidencefor a functional interaction between the 530 loop of the ribosome andRF1 that is consistent with, but not predicted by, the 5.9 Å X-raystructure of RF1 bound to the ribosome³⁵. In contrast to temperaturesensitive mutants in the essential RF1 protein, mutants in the cellularribosome that allow transient increases in suppression across all mRNAsat the expense of cellular viability^(33, 36, 37), or mutants in rRNAthat cause misreading of amber codons in the absence of a cognate tRNA³⁸the effect of ribo-X is localized to the population of orthogonal mRNAsthat it decodes. Ribo-X increases tRNA dependent amber suppression in atarget gene without increasing read through of amber codons in naturalcellular mRNAs and therefore does not perturb decoding of the naturaltranscriptome.

Ribo-X is Optimized for the 32-38 Pair in Extended Anticodon tRNAs

While we observe a strong effect on UAGN and UAG decoding we also seeimprovements, albeit more modest, in the efficiency of decoding thequadruplet codons AGGA and CCCU codons by cognate extended anticodontRNAs (FIG. 4). In these cases release factors are not believed tocompete with the tRNA for A-site binding, suggesting that the decreasedfunctional interaction with RF1 cannot account for all the properties ofribo-X. We therefore looked for features within the tRNA structure whichribo-X might be optimized to recognize.

The extended anticodon tRNAs used in this study have purines at position32 in the anticodon loop and phylogenetically unusual combinations ofnucleotides at positions 32 and 38³⁹. Uhlenbeck and coworkers haveshown, in the context of triplet decoding, that natural variation in theidentity of the 32-38 pair can influence the affinity of tRNAs for theribosomal A-site⁴⁰. The A site of ribo-X may have optimized affinity forexpanded anticodon tRNAs with unusual nucleotides at position 32 and 38while maintaining close to optimal affinity for natural tRNAs.Consistent with this view, we find that conversion of the 32-38 pair inthe AGGA tRNA from A32C38 to C32A38, as found in tRNA^(ser2) produces anextended anticodon tRNA read equally poorly by the progenitorO-ribosomes and by ribo-X (FIG. 4).

We have demonstrated the first example of synthetic evolution ofribosome function in living cells. We have shown that orthogonalribosomes can be evolved to more efficiently decode a range of extendedcodons using tRNAs with extended anticodon loops. The evolved orthogonalribosome, ribo-X, preferentially reads quadruplet codons with extendedanticodon tRNAs and can show specificity for Watson-Crick base pairs atthe fourth position of the codon-anticodon interaction. Ribo-X alsoimproves amber suppression by amber suppressor tRNAs. Finally we haveprovided experimental support for a model which explains the mode ofaction of ribo-X, and implicates the 530 loop, in the ribosome decodingcentre, in functional interactions with RF1.

The observation that ribo-X can improve the efficiency with which fulllength proteins are synthesized from orthogonal mRNAs containing ambercodons has technological significance. Almost all current methods ofintroducing unnatural amino acids into proteins in vivo rely on ambersuppression, and produce a large fraction of truncated protein⁴¹⁻⁴⁴, asa result of release factor mediated peptide chain termination. Releasefactor mediated protein truncation reduces protein yield and, for invivo studies using unnatural amino acids to probe cellularfunction^(45, 46), truncated proteins can have unforeseen functional andphenotypic effects that perturb precisely the system underinvestigation. Ribo-X should facilitate in vivo studies with unnaturalamino acids and may also allow improvements in total protein yield forexpression systems that exploit incorporation of unnatural amino acidsin response to the amber codon. Importantly since ribo-X's primary modeof action on UAG codons is decreasing release factor binding it willexert its enhancement of UAG decoding for the wide range of ambersuppressor tRNAs used for unnatural amino acid incorporation in E. coli.

By improving the quadruplet decoding of the orthogonal ribosome we havediverged the decoding properties of the orthogonal ribosome from thoseof the cellular ribosome such that the same insertion signal is readwith a different efficiency on cellular and orthogonal mRNAs within thesame cell. Since the meaning of codons on any mRNA is set by thetranslational machinery that decodes that mRNA, it may be possible touse extensions of our approach to bypass the “frozen accident” of theexisting genetic code⁴⁷ and write quadruplet codes or other geneticcodes on orthogonal mRNAs, using tRNAs that are poor substrates for thecellular ribosome but are efficiently decoded by evolved orthogonalribosomes. Such parallel and independent codes (orthogonal geneticcodes) would expand the information storage capacity of the cell andmight be used to extend in vitro computation^(48, 49) to living cells.Moreover orthogonal codes might be used to encode the biosynthesis ofunnatural polymers and to create insulated genetic codes for encodingthe components of synthetic genetic circuits in a form unreadable by,and therefore not functionally transmissible to, natural biologicalentities.

Example 3 Evolved Orthogonal Ribosomes with Enhanced Efficiency

Design of a Ribosome Decoding Centre Library

The A-site of the ribosome is the gateway to the tRNA translocationcorridor composed of the A, P and E sites²⁸⁻³⁰. In response to an ambercodon in the A-site of the ribosome RF-1 can bind to the A-site andcompete with decoding of amber suppressor tRNAs. We reasoned thatcombinations of mutations in 16S rRNA, which forms the A-site of theribosome, might yield a variant A-site that allows amber suppressortRNAs to compete more effectively with RF-1 for A-site binding, and thusfavour amber suppression and elongation over polypeptide chaintermination.

To design an A-site library (FIG. 8) we examined the structures of tRNAsor RF-1 bound to the ribosomal A-site²⁹⁻³². These structures show thatthe 530 loop in 16S rRNA is proximal to both substrates (FIG. 8). Wereasoned that combinations of mutations in the 530 loop might maintaintRNA binding, but decrease functional interaction with RF-1 in thepresence of a UAG codon. We therefore randomized seven nucleotides(529-535) in the 530 loop to all combinations of nucleotides to createan N7 library. Moreover, we created longer and shorter 530 loop sequencelibraries (N5, N6, N8, and N9 (FIG. 8)) to expand the functional spacesampled. All libraries were more than 99% complete (Supplementary Table1).

Selection of Evolved Decoding in Orthogonal Ribosomes

To create a selection system for orthogonal ribosomes that moreefficiently read amber codons, we required a reporter of orthogonalribosome activity that contains selector codons. We decided to workinitially with a UAGA containing reporter and tRNA^(ser2) (UCUA) (FIG.14), which is aminoacylated by seryl-tRNA synthetase³³, rather than asimple UAG suppressor, because it allows selection for improved ribosomeactivity over a larger dynamic range. Cells containing O-cat (UAGA103,UAGA146)/tRNA^(ser2) (UCUA) and the O-ribosome had an IC₅₀ onchloramphenicol of 25 μg ml⁻¹. For comparison, the O-cat reporter devoidof UAGA codons supports growth on 500 μg ml⁻¹ of chloramphenicol in thepresence of the O-ribosome.

To select mutant ribosomes that more efficiently decode the UAGA codonwe combined each orthogonal ribosome library with O-cat (UAGA103,UAGA146)/tRNA^(ser2) (UCUA) (in which cat containing UAGA codons istranslated from an orthogonal ribosome binding site, FIG. 15) andchallenged the cells to grow on chloramphenicol concentrations at whichthe O-ribosome does not support growth. No clones containing insertionsor deletions survived for libraries N5, N6, N8 and N9, suggesting thatthe 530 loop is intolerant to longer or shorter sequences of anycomposition. However, clones from the N7 library did survive on 100 μgml⁻¹ chloramphenicol. All ten clones sequenced from the N7 selectionwere identical, and contained the mutations U531G and U534A in 16S rRNA(FIG. 9). The U531G mutation is present in only two sequenced vertebratemitochondrial rRNAs while the U534A mutation is present in 0.2% ofbacterial rRNAs. No sequenced natural ribosome contains this combinationof mutations³⁴. Because it remained a formal possibility that moreefficient ribosomes for UAG decoding existed in the libraries but werenot captured by the UAGA selection we repeated the selection withreporters containing the UAG codon and an amber suppressor derived fromtRNA^(ser2) (FIG. 14). We found that the same sequence was uniquelyselected. We therefore decided to characterize the U531G, U534A mutantribosome, which we refer to as ribo-X, in more detail.

Ribo-X Enhances tRNA Dependent UAGA Decoding

To measure the extent to which ribo-X enhances tRNA^(ser2) (UCUA)dependent decoding of the UAGA codon, we compared the chloramphenicolresistance of cells containing O-cat (UAGA103, UAGA146)/tRNA^(ser2)(UCUA) and either ribo-X or the progenitor O-ribosome (FIG. 9). Ribo-Xcontaining cells survive on concentrations of chloramphenicol five timeshigher than cells containing the progenitor O-ribosome. The enhanced,tRNA^(ser2) (UCUA) dependent, quadruplet decoding was further confirmedby in vitro chloramphenicol acetyl transferase (CAT) assays³⁵ (FIG. 9).Similar ribo-X mediated enhancements were observed using tRNA^(ser2)(CUA) and a cognate O-cat reporter. The chloramphenicol resistanceconferred on cells by ribo-X and O-cat and the progenitor O-ribosome andO-cat is identical (500 μg ml⁻¹), indicating that ribo-X is efficientand processive in translation of sense codons.

Ribo-X and Natural Ribosomes have Comparable Fidelity

To demonstrate that ribo-X synthesizes proteins with a fidelitycomparable to the natural ribosome, we compared the mass spectra, andamino acid mis-incorporation frequency of proteins synthesized bywild-type ribosomes, the progenitor orthogonal ribosome and ribo-X.

Expression of O-gst-malE (a genetic fusion between the genes encodingglutathione-S-transferase (GST) and maltose binding protein (MBP) drivenby an orthogonal ribosome binding site) in the presence of ribo-X or theprogenitor O-ribosome produced GST-MBP with a purified yield of 30-40 mgl⁻¹, comparable to the yield of GST-MBP produced from a gst-malE fusionby wild-type ribosomes. As expected, no GST-MBP can be purified fromO-gst-malE in the absence of orthogonal ribosomes (FIG. 10 a). Thrombincleavage of GST-MBP, at a site between GST and MBP in the protein fusion(FIG. 16), produced two proteins that were amenable to massdetermination by electrospray ionization mass-spectrometry. Proteinsproduced from each ribosome had the same mass (FIG. 10 b). To explicitlycompare the translational fidelity of ribo-X to that of progenitorribosomes, we measured the frequency of ³⁵S-cysteine mis-incorporation³⁶into MBP, which contains no cysteine codons (FIG. 10 c). The errorfrequency per codon translated by ribo-X was less than 1×10⁻³. Controlexperiments with the progenitor orthogonal ribosome and the wild-typeribosome allowed us to put the same limit on their error frequency. Thislimit compares well with a previous measurement for amino acidmis-incorporation frequency, as measured by ³⁵S-cysteinemis-incorporation³⁶, of 4×10⁻³ errors per codon. To further probe thetranslational fidelity of ribo-X with respect to defined perturbationsin the codon-anticodon interaction, we took advantage of a dualluciferase reporter system (DLR) that has previously been used tomeasure the fidelity of natural and error-prone ribosomes in decodingnear-cognate and non-cognate codons³⁷. We created a DLR with anorthogonal ribosome-binding site (O-DLR), and demonstrated that itstranslation is dependent on the presence of a cognate orthogonalribosome (FIG. 17). We translated O-DLR variants, for which K529(AAA)was mutated at each position of the codon-anticodon interaction (FIG. 10d), using ribo-X or the progenitor orthogonal ribosome, and compared theresulting luciferase activities as a measure of translationalmis-reading. We find that the fidelity of ribo-X is at least as good asthat measured for the progenitor orthogonal ribosome and the naturalribosome across all four codon-anticodon interactions tested.

Overall, the mass spectra, ³⁵S mis-incorporation assay and dualluciferase assays demonstrate that ribo-X has a translational fidelitycomparable to that of the natural ribosome.

Increased Efficiency Unnatural Amino Acid Incorporation

To demonstrate the substantial increase in efficiency of site-specificunnatural amino acid incorporation with ribo-X, we chose to work withthe photocrosslinking amino acid p-benzoyl-L-phenylalanine (Bpa)³⁸. Thisamino acid has been added to the genetic code of E. coli, yeast andmammalian cells and used extensively to map the topology ofprotein-protein interactions in vitro and in vivo^(5, 20, 39-42).

We expressed gst(UAG)malE in the presence of ap-benzoyl-L-phenylalanyl-tRNA synthetase/tRNA_(CUA) (BpaRS/tRNA_(CUA))pair³⁹ (evolved from the MjTyrRS/tRNA_(CUA) pair), Bpa and wild-typeribosomes (FIG. 11 a). As expected this produced GST-MBP, incorporatingBpa, with low efficiency (24%). However when we synthesized GST-MBPcontaining Bpa from O-gst(UAG)malE using the BpaRS/tRNA_(CUA) pair, Bpa,and ribo-X, the efficiency increased to 62%. As expected, based on thepreviously reported specificity of BpaRS, full-length protein synthesisis Bpa dependent³⁹. In our experiments, performed in Luria Bertani (LB)medium as previously described²³, we see a small amount of full-lengthprotein synthesis that is BpaRS and tRNA_(CUA) dependent, but not aminoacid dependent (compare FIG. 11 a, lanes 2 and 6 and 10). This effect isminimized in minimal medium, where the total yields of overexpressedproteins are also approximately five times lower (FIG. 18). In thepresence of Bpa, the aminoacylation of natural amino acids ontotRNA_(CUA) by non-cognate aminoacyl-tRNA synthetases observed in richmedia is out-competed, and incorporation of Bpa is quantitative²³ (FIG.11 b, c, d). Mass spectrometry shows that BpaRS expression from pSupBpadoes not lead to detectable levels of unnatural amino acid incorporationin response to sense codons (via misacylation of endogenous tRNAs), asexpected from the observation that MjTyrRS does not aminoacylate any E.coli tRNAs with tyrosine, even in the absence of competing endogenousaminoacyl-tRNA synthetase enzymes⁴³. In the absence of a functionalaminoacyl-tRNA synthetase/tRNA_(CUA) pair ribo-X terminates translationon the amber codon, and no full-length GST-MBP fusion is purified (FIG.11 a, lane 10). Similarly, ribo-X does not measurably enhanceread-through of a UAA or UGA codons.

The ribo-X mediated enhancement of efficiency was even more dramatic fora gene containing two amber stop codons (FIG. 11 a): wild-type ribosomesproduced GST-MBP containing two Bpas from gst(UAG)₂malE with anefficiency of less than 1%, while ribo-X produced GST-MBP containing twoBpas with an efficiency at least twenty-fold higher (22%) fromO-gst(UAG)₂malE. Extrapolation of the single UAG efficiencies to twosites predicts efficiencies of 38% and 6% for ribo-X and the wild-typeribosome respectively. Comparison of the ratio of predicted to observedefficiencies for each ribosome suggests that ribo-X may be more robustthan the wild-type ribosome to context effects which decrease theefficiency of UAG suppression⁴⁴ Electrospray ionization massspectrometry of MBP produced by ribo-X in the presence of theBpaRS/tRNA_(CUA) pair and Bpa confirmed the incorporation of 2 Bpas; nopeaks were detected corresponding to the incorporation of natural aminoacids (FIG. 11 b). The sites of Bpa incorporation were further confirmedby analysis of the MS/MS fragmentation series of the relevantchymotryptic peptides (FIGS. 11 c & 11 d). We observe that the ribo-Xmediated improvement in efficiency for one and two amber codons isconserved in minimal medium (FIG. 18), demonstrating that the effectmediated by ribo-X is robust under different expression conditions.Overall, the protein expression data and mass spectrometry data clearlydemonstrate that the modular combination of ribo-X, BpaRS/tRNA_(CUA) andan orthogonal mRNA containing multiple UAG codons allows thesite-specific incorporation of Bpa with high fidelity and efficiency atmultiple sites in GST-MBP.

Materials and Methods to Example 3:

Construction of Ribosome Libraries and Reporters

16S rDNA libraries were constructed by enzymatic inverse PCR²⁵ on pRSFvectors containing a previously described O-rDNA (pRSF-O-rDNA)⁴⁸. Tocreate the UAGA reporter plasmid we introduced the amber derived UAGAcodon at two sites in the chloramphenicol acetyl transferase (cat) gene(Ser103 and Ser146, an essential and conserved catalytic serineresidue⁴⁹ that ensures the fidelity of incorporation, FIG. 15),downstream of an orthogonal ribosome-binding site, producing O-cat(UAGA103, UAGA146). This construct was created by multiple rounds ofQuik Change mutagenesis (Stratagene) on an O-cat reporter derived fromp21²⁵ by replacement of the cat-upp fusion with the cat gene alone. tRNAgenes were introduced into the O-cat (UAGA103, UAGA146) plasmid at aunique Bst Z17I restriction site, via a cassette containing a 5′synthetic lpp promoter and a 3′ rrnC transcriptional terminator, tocreate the vector O-cat (UAGA103, UAGA146)/tRNA(UCUA); the sequence ofthe extended anticodon is written 5′ to 3′. UAG codon reporters and CUAanticodon tRNAs were derived by Quik Change mutagenesis from the UAGA orUCUA constructs. All final plasmids were confirmed by DNA sequencing.For a complete description of oligonucleotides used for vectorconstruction see Supplementary Table 2.

Selection of Evolved O-Ribosomes

To select O-ribosomes with improved UAGA decoding, each pRSF-O-rDNAlibrary was transformed by electroporation into GeneHog E. coli(Invitrogen) containing O-cat (UAGA103, UAGA146)/tRNA(UCUA). Transformedcells were recovered for 1 h in SOB medium containing 2% glucose andused to inoculate 200 ml of LB-GKT (LB medium with 2% glucose, 25 μgml⁻¹ kanamycin and 12.5 μg ml⁻¹ tetracycline). After overnight growth(37° C., 250 r.p.m., 16 h), 2 mL of the cells were pelleted bycentrifugation (3000 g), and washed three times with an equal volume ofLB-KT (LB medium with 12.5 μg ml⁻¹ kanamycin and 6.25 μg ml⁻¹tetracycline). The resuspended pellet was used to inoculate 18 ml ofLB-KT, and the resulting culture incubated (37° C., 250 r.p.m. shaking,90 min). To induce expression of plasmid encoded O-rRNA, 2 ml of theculture was added to 18 ml LB-IKT (LB medium with 1.1 mMisopropyl-D-thiogalactopyranoside (IPTG), 12.5 μg ml⁻¹ kanamycin and6.25 μg ml⁻¹ tetracycline) and incubated for 4 h (37° C., 250 r.p.m.).Aliquots (250 μl optical density at 600 nm (OD₆₀₀)=1.5) were plated onLB-IKT agar (LB agar with 1 mM IPTG, 12.5 μg ml⁻¹ kanamycin and 6.25 μgml⁻¹ tetracycline) supplemented with 50 μg ml⁻¹ chloramphenicol andincubated (37° C., 40 h).

Characterization of Evolved O-Ribosomes

To separate selected pRSF-O-rDNA plasmids from the O-cat (UAGA103,UAGA146)/tRNA^(ser2) (UCUA) reporter plasmids, total plasmid DNA fromselected clones was purified and digested with Not I restrictionendonuclease, and transformed into DH10B E. coli. Individualtransformants were replica plated onto kanamycin agar and tetracyclineagar and plasmid separation of pRSF-O-rDNA from the reporter confirmedby restriction digest and agarose gel analysis.

To quantify the UAGA decoding activity of selected 16S rDNA clones,selected pRSF-O-rDNA plasmids were co-transformed with O-cat (UAGA103,UAGA146) or O-cat (UAGA103, UAGA146)/tRNA^(ser2) (UCUA). Cells wererecovered (SOB, 2% glucose, 1 h) and used to inoculate 10 mL of LB-GKT,which was incubated (16 h, 37° C., 250 r.p.m.). 1 ml of the resultingculture was used to inoculate 9 ml of LB-KT, which was incubated (90min, 37° C., 250 r.p.m.). 1 ml of the LB-KT culture was used toinoculate 9 ml of LB-IKT medium, which was incubated (37° C., 250r.p.m., 4 h). Individual clones were transferred to a 96-well block andarrayed, using a 96 well pin tool, onto LB-IKT agar plates containingchloramphenicol at concentrations from 0 to 250 μg ml⁻¹. The plates wereincubated (37° C., 16 h). We performed analogous experiments for othertRNA codon pairs.

To extract soluble cell lysates for in vitro CAT assays, 1 ml of eachinduced LB-IKT culture was pelleted by centrifugation at 3,000 g. Thecell pellets were washed three times with 500 μl Washing Buffer (40 mMTris-HCl, 150 mM NaCl, 1 mM EDTA, pH 7.5) and once with 500 μl LysisBuffer (250 mM Tris-HCl, pH 7.8). Cells were lysed in 200 μl LysisBuffer by five cycles of flash-freezing in dry-ice/ethanol followed byrapid thawing in a 50° C. water-bath. Cell debris was removed from thelysate by centrifugation (12,000 g, 5 min) and the top 150 μl ofsupernatant frozen at −20° C. To assay CAT activity in the lysates, 10μl of soluble cell extract was mixed with 2.5 μl of FAST CAT Green(deoxy) substrate (Invitrogen) and pre-incubated (37° C., 5 min). 2.5 μlof 9 mM acetyl-CoA (Sigma) was added, and the reaction incubated (37°C., 1 h). The reaction was stopped by the addition of ice-cold ethylacetate (200 μl, vortex 20 s). The aqueous and organic phases wereseparated by centrifugation (12,000 g, 10 min) and the top 100 μl of theethyl acetate layer collected. 1 μl of the collected solution wasspotted onto a silica gel TLC plate (Merck) for thin-layerchromatography in chloroform:methanol (85:15 v/v). The fluorescence ofthe spatially resolved substrate and product was visualized andquantified using a phosphorimager (Storm 860, Amersham Biosciences) withexcitation and emission wavelengths of 450 nm and 520 nm, respectively.

Construction of GST-MBP Protein Expression Vectors

gst was amplified from pGEX-2T (GE Healthcare) with the primers:GAACTCGAGACAATTTTCATATCCCTCCGCAAATGTCCCCTATACTAGGTTATTGGA AAATTAAG (SEQID NO:1) and GAAGAGGTACCCGTCACGATGAATTCCCGGGGATCCACGCGGAAC (SEQ IDNO:2), and digested with Xho I and Kpn I. malE was amplified from pMAL(NEB) with PCR primers GAAGGGTACCTCAAAATCGAAGAAGGTAAACTGGTAATC (SEQ IDNO:3) and CCAAAGCTTAGCTTGCCTGCAGGTCGACTC (SEQ ID NO:4) and digested withHind III and Kpn I. pO-gst-malE was generated from pGFPmut3.1 (Promega),by replacing A¹⁹¹T¹⁹²A¹⁹³ in the vector with CTCGAG (Xho I site). Thismutates the lac operator and renders expression of the downstream geneconstitutively active. Gfp was excised from between the Hind III siteand the newly introduced Xho I site, and the gst-malE fusion introducedwith the same sites via a three-fragment ligation. The vector p gst-malEwas created by changing the orthogonal ribosome binding site to a singlewildtype ribosome binding site with the enzymatic inverse PCR primers:GTAGGTCTCGGATCCCCGGGTACCTAGAATTAAAGAGGAGAAATTAAGCATGTCCCCTATACTAGGTTATTG (SEQ ID NO:5) andGTAGGTCTCGGATCCTCTAGAGTCGACCTGCAGGAATGCAAGCTTGGCGTAACTCGAGCCGCTCACAATTCCACAC (SEQ ID NO:6). To create vectors containing asingle amber codon between gst and malE (pgst(UAG)malE andpO-gst(UAG)malE) the Tyr codon, TAC, in the linker between gst and malEwas changed to TAG by Quik Change mutagenesis (Stratagene), using theprimers GAATTCATCGTGACGGGTAGCTCAAAATCGAAGAAGGTAAACTGGTAATCTG (SEQ IDNO:7) and CTTCGATTTTGAGCTACCCGTCACGATGAATTCCCGGGGATCCACGCGGAAC (SEQ IDNO:8). For double UAG mutants we additionally mutated the fourth codonin malE from GAA to TAG by Quik Change, with the primersCTCAAAATCTAGGAAGGTAAACTGGTAATCTGGATTAACGGCGATAAAG (SEQ ID NO:9) andCAGTTTACCTTCCTAGATTTTGAGCTACCCGTCACGATG (SEQ ID NO:10) to create thevectors pgst(UAG) ^malE and pO-gst(UAG) 2malE.

Construction of P1P2 Ribosomes for Protein Expression

The kanamycin resistance gene and the SC101* origin of plasmidpZS*24-MCS1⁵° were amplified using the following primers: KanSC101fw,ACT GGA TCC TGC TAG AGG CAT CAA ATA AAA C (SEQ ID NO:11), andKanSC101rv, AGT ACC GGT TAG ACG TCG GAA TTG CCA GC (SEQ ID NO:12). Theresulting PCR product was digested with Barn HI and Age I. The rrnBoperon, including the P1P2 promoter and rrnC terminator, was excisedfrom plasmid pTrc P1P2 rrnB by digestion with NgoM IV and Barn HI, andthe amplified SC101* fragment and PIP2rrnB fragment were ligated tocreate plasmid pSC101*-BD. The Xho I/Xba I fragment of this plasmid wasreplaced with a corresponding fragment from pTrcRSF-O-rRNA orpTrcRSF-ribo-X, yielding pSC101*-O-ribosome and pSC101*-ribo-X,respectively.

Expression and Purification of GST-MBP Fusions

E. coli containing the appropriate plasmid combinations were pelleted(3000 g, 10 min) from 50 ml, overnight cultures, resuspended in 1 mlLysis buffer (Phosphate buffered saline (PBS) supplemented with 1×protease inhibitor cocktail (Roche), 1 mM PMSF and 1 mg ml⁻¹ lysozyme(Sigma)), and incubated (15 min, 37° C., 1000 r.p.m.). Cells werechilled on ice before lysis by sonication (30 s, 30 W). The lysate wasclarified by centrifugation (6 min, 25000 g, 2° C.). GST containingproteins from the lysate (875 μg, 400 μl) were bound in batch (1 h, 4°C.) to 50 μl of Glutathione sepharose beads (GE Healthcare). Beads werewashed 3 times with 1 ml PBS, before elution by heating for 10 min at80° C. in 60 μl 1×SDS gel-loading buffer. All samples were analysed on4-20% Tris-Glycine gels (Invitrogen).

The densities of the bands for GST-MBP and GST were quantified fromCoomassie stained gels with NIH image 1.63. We divided thebackground-corrected values by the molecular mass of the correspondingproteins (GST-MBP, 71 kDa and GST, 27 kDa) and used these values tocalculate the percentage of amber codon suppression, by dividing theamount of GST-MBP by total amount of protein.

³⁵S-Cysteine Mis-Incorporation

GeneHog E. coli containing either pO-gst-malE and pSC101*-O-ribosome,pO-gst-malE and pSC101*-ribo-X or pgst-malE were resuspended in LB media(supplemented with ³⁵S-cysteine (1000 Ci mmol⁻¹) to a finalconcentration of 3 nM, 750 μM methionine, 25 μg ml⁻¹ ampicillin and 12.5μg ml⁻¹ kanamycin) to an OD₆₀₀ of 0.1, and cells were incubated (3.5 h,37° C., 250 r.p.m.). 10 mL of the resulting culture was pelleted (5000g, 5 min), washed twice (1 mL PBS per wash), resuspended in 1 mL PBScontaining 1% Triton-X, and lysed on ice by pipetting up and down. Theclarified cell extract was bound to 100 μL of glutathione sepharosebeads (1 h, 4° C.) and the beads were pelleted (5000 g, 10 s) and washedtwice in 1 mL PBS. The beads were added to 10 mL polypropylene column(Biorad) and washed (30 mL of PBS; 10 mL 0.5M NaCl, 0.5×PBS; 30 mL PBS)before elution in 1 mL of PBS supplemented with 10 mM glutathione.Purified GST-MBP was digested with 12.5 units of thrombin, to yield aGST fragment and an MBP fragment. The reaction was loaded onto anSDS-PAGE gel to resolve the GST, MBP and thrombin, and stained withGelCode blue (Invitrogen). The ³⁵S activity in the GST and MBP proteinbands were quantified by densitometry, using a Storm Phosphoimager(Molecular Dynamics) and ImageQuant (GE Healthcare) and by scintillationcounting of excised bands. The error frequency per codon for eachribosome examined was determined as follows: GST contains 4 cysteinecodons, so the number of counts per second (cps) resulting from GSTdivided by four gives A, the cps per quantitative incorporation ofcysteine. MBP contains no cysteine codons, but mis-incorporation atnon-cysteine codons gives B cps. Since GST and MBP are present inequimolar amounts, (A/B)×410, where 410 is the number of amino acids, inthe MBP containing thrombin cleavage fragment, gives the number of aminoacids translated for one cysteine mis-incorporation C. Assuming themis-incorporation frequency for all 20 amino acids is the same as thatfor cysteine the number of codons translated per mis-incorporation isC/20, and the error frequency per codon is given by (C/20)⁻¹.

Dual Luciferase Assays

pO-DLR contains a genetic fusion between a 5′ Renilla Luciferase (R-luc)and a 3′ Firefly Luciferase (F-luc) on an orthogonal ribosome bindingsite. To create pO-DLR the R-luc open reading frame from the plasmidpGL4.70[hRluc] (Promega) was amplified by PCR using the primersGAACTCGAGGGCGCGGCTTTCATATCCCTCCGCAAATGGCCTCCAAGGTGTACGACCCCGAGCAACGCAAACGCATG (SEQ ID NO:13) andGCTAGATCTCCTAGGGGCCCCCGTCGAGATTTGCTCGTTCTTCAGCACGCGCTCC ACGAAGCTC (SEQID NO:14). The PCR product was digested with Xho I and Bgl II. The F-lucORF was amplified with primer pairAGGAGATCTAGCGCTGGATCCCCCGGGGAGCTCATCGAAGATGCCAAAAACATTA AGAAGGGCCCAG(SEQ ID NO:15) and GACAAGCTTACACGGCGATCTTGCCGCCCTTCTTG (SEQ ID NO:16)and digested with Bgl II and Hind III. The gst-malE gene fusion wasexcised from pO-gst-malE by Xho I and Hind III digestion and pO-DLRcreated by a triple ligation of the released vector backbone with thedigested F-luc PCR product and the digested R-luc PCR product. Pwt-DLRwas created by a similar strategy, but using the primer pairGAACTCGAGTACCTAGATATAAAGAGGAGAAATTAAGCATGGCCTCCAAGGTGTACGACCCCGAGCAACGCAAACGCATG SEQ ID NO:17) andGCTAGATCTCCTAGGGGCCCCCGTCGAGATTTGCTCGTTCTTCAGCACGCGCTCC ACGAAGCTC (SEQID NO:18) to amplify the R-luc ORF. Codon 529 variants were created byQuik Change Mutagenesis (Stratagene).

pO-DLR, and its K529 codon variants, were transformed into GeneHog E.coli cells with pSC101*-O-ribosome or pSC101*-ribo-X. pwt-DLR, and itsK529 codon variants, were transformed into GeneHog cells withpSC101*-BD. Individual colonies were incubated (37° C., 250 r.p.m., 20h) in 2 mL LB supplemented with ampicillin (100 μg ml⁻¹) and kanamycin(50 μg ml⁻¹), pelleted (5000 g, 5 min) and resuspended in 200 μL (1 mgml⁻¹ lysozyme, 10 mM Tris (pH 8.0), 1 mM EDTA). Cells were incubated onice for 20 min, frozen on dry ice, and thawed on ice. 10 μL samples ofthis extract were assayed for firefly (F-luc) and Renilla (R-luc)luciferase activity using the Dual-Luciferase Reporter Assay System(Promega). Each ribosome reporter combination was assayed from fourindependent cultures using an Orion microplate luminometer (BertholdDetection Systems) and the data analyzed as previously described. Theerror reported is the standard deviation.

Mass Spectrometry

25 μM GST-2BPA-MBP (GST-MBP with Bpas incorporated in response to twoamber codons) in 22 μl 40 mM (NH₄)HCO₃ was alkylated and digested withchymotrypsin overnight. To obtain a fragment series for the N-terminalBpa incorporation, 5 μl of a tenfold dilution of the chymotrypticpeptide mixture was desalted and concentrated by using a GELoader tipfilled with Poros R3 sorbent (Perseptive Biosystems). The bound peptideswere eluted with 1 μl of 40% acetonitrile/4% formic acid directly into ananospray capillary and then introduced into an API QSTAR pulsar ihybrid quadrupole-time-of-flight mass spectrometer (MDS Sciex). Production scans were carried out in positive ion-mode and MS survey scans forpeptides measured. Selected ions (m/z=668.4⁴⁺) were fragmented bycollision-induced dissociation (CID) with nitrogen in the collision celland spectra of fragment ions produced were recorded in thetime-of-flight mass analyzer. To obtain a fragment series for theC-terminal Bpa incorporation, peptides from the chymotryptic digest wereseparated by nanoscale liquid chromatography (LC Packings, Amsterdam,The Netherlands) on a reverse-phase C18 column (150×0.075 mm internaldiameter, flow rate 0.25 ml min⁻¹). The eluate was introduced directlyinto a Q-STAR hybrid tandem mass spectrometer (LC-MS/MS) and peptidewith m/z=469.7⁴⁺ fragmented.

Protein total mass was determined on an LCT time-of-flight massspectrometer with electrospray ionization (ESI). (Micromass). Proteinswere re-buffered to 10 mM ammonium bicarbonate pH 7.5 and diluted 1:100into 50% methanol, 1% formic acid. Samples were infused into the ESIsource at 10 ml min⁻¹, using a Harvard Model 22 infusion pump (HarvardApparatus) and calibration performed in positive ion mode using horseheart myoglobin. 60-80 scans were acquired and added to yield the massspectra. Molecular masses were obtained by deconvoluting multiplycharged protein mass spectra using MassLynx™ version 4.1 (Micromass).Theoretical molecular masses of wild type proteins were calculated usingProtpram, and theoretical masses for unnatural amino acid containingproteins adjusted manually.

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All patents, patent applications, and published references cited hereinare hereby incorporated by reference in their entirety. While thisinvention has been particularly shown and described with references topreferred embodiments thereof, it will be understood by those skilled inthe art that various changes in form and details may be made thereinwithout departing from the scope of the invention encompassed by theappended claims.

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The invention claimed is:
 1. An evolved orthogonal ribosomal RNA (rRNA)which possesses an enhanced efficiency of tRNA-dependent reading oforthogonal mRNA codons, wherein said orthogonal rRNA is a 16S rRNAcontaining a mutated 530 loop such that the 16S rRNA comprises thesequence 529-GGGAAAA-535.
 2. An evolved orthogonal rRNA according toclaim 1, wherein the orthogonal mRNA codons are extended codons or stopcodons.
 3. An evolved orthogonal rRNA according to claim 2, wherein theorthogonal mRNA codon is a quadruplet codon or an amber stop codon. 4.An evolved orthogonal rRNA according to claim 1, which possesses adecreased functional interaction with release factor 1 (RF-1).
 5. Anorthogonal ribosome incorporating an evolved orthogonal rRNA accordingto claim
 1. 6. A cell comprising two or more protein translationmechanisms, wherein: (a) a first mechanism is the natural translationmechanism wherein mRNA is translated by a ribosome in accordance withthe natural genetic code; and (b) a second mechanism is an artificialmechanism, in which orthogonal mRNA comprising orthogonal codons istranslated by an orthogonal ribosome; wherein the orthogonal codons inthe orthogonal mRNA are (i) not translated by the natural ribosome, or(ii) translated more efficiently by the orthogonal ribosome than by thenatural ribosome, or (iii) translated into different polypeptides by theorthogonal ribosome and the natural ribosome, and wherein the orthogonalribosome incorporates an evolved orthogonal ribosomal RNA (rRNA) whichpossesses an enhanced efficiency of tRNA-dependent reading of orthogonalmRNA codons, wherein said orthogonal rRNA is a 16S rRNA containing amutated 530 loop such that the 16S rRNA comprises the sequence529-GGGAAAA-535.